1. Field of the Invention
This invention relates to the field of polypeptide ligand and receptor interactions. In particular, it relates to the field of selecting and screening antagonists and agonists for polypeptide ligands.
2. Description of the Background Art
Ligand induced receptor oligomerization has been proposed as a mechanism of signal transduction for the large family of tyrosine kinase receptors that contain an extracellular ligand binding domain (for reviews see Yarden, Y., et al., Ann. Rev. Biochem 57:443-478 (1988); Ullrich, A., et al., Cell 61:203-212 [1990]). In these models binding of one hormone molecule (or subunit) (H) per receptor (R) is thought to induce formation of an H2R2 complex. For example, crosslinking and non-dissociating electrophoretic studies suggest that epidermal growth factor (EGF) promotes dimerization of the EGF receptor followed by receptor autophosphorylation and activation of the intracellular tyrosine kinase (Shector, Y., et al., Nature 278:835-838 (1979); Schreiber, A. B., et al., J. Biol. Chem., 258: 846-853 (1983); Yarden, Y., et al., Biochemistry, 26:1434-1442 (1987); Yarden, Y., et al., Biochemistry 26:1443-1451 (1987). Studies of other tyrosine kinase receptors including the insulin receptor (Kahn, C. R., et al., Proc. Natl. Acad. Sci. USA 75:4209-4213 (1978); Kubar J., et al., Biochemistry 28:1086-1093 (1989); Heffetz, D., et al., J. Biol. Chem. 261:889-894 (1986), platelet derived growth factor (PDGF) receptor (Heldin, C. H., et al., J. Biol. Chem. 264:8905-8912 (1989); Hammacher, A., et al., EMBO J. 8:2489-2495 (1989); Seifert, R. A., et al.,J. Biol., Chem. 264:8771-8778 (1989)) and insulin-like growth factor (IGF-I) receptor (Ikari, N., et al., Mol. Endocrinol. 2:831-837.), indicate that oligomerization of the receptor is tightly coupled to the biological effect. Other groups have recently crystallized a polypeptide hormone in complex with its extracellular binding domain (Lambert, G., et al., J. Biol. Chem. 264:12730-12736 (1989); Gunther, N., et al., J. Biol. Chem. 265:22082-22085 (1990)). However, more detailed analyses of the structural perturbations and requirements for ligand induced changes in these or other receptors have been hampered because of the complexities of these membrane associated systems and the lack of suitable quantities of highly purified natural or recombinant receptors.
When purified receptors were available the assay procedures were often structured so that the nature of the hormone-receptor complex was not recognized. In U.S. Pat. No. 5,057,417, hGH binding assays were conducted using 125I-hGH competition with cold hGH for binding to the extracellular domain of recombinant hGH receptor (hGHbp), or hGH binding protein; the resulting complex was treated with antibody to the hGHbp, plus polyethylene glycol, to precipitate the complex formed. These immunoprecipitation assays suggested that hGH formed a 1:1 complex with hGHbp. This immunoprecipitation assay correctly detected the amount of 125I-hGH bound, but it incorrectly indicated a 1:1 molar ratio.
Various solid phase assays for hGH receptor and binding protein have been used. Such assays detected the amount of hGH bound but not the molar ratio of hGH to receptor. Binding assays with solid phase or with membrane fractions containing hGH receptor were not suitable for determining the molar ratio of hGH to receptor due to an inability to detect the total amount of active receptor and/or the amount of endogenous hGH bound. Based upon earlier work, such as with EGF, the art assumed the hGH-receptor complex would be an H2R2 tetramer.
The hGH receptor cloned from human liver (Leung, D. W. et al., Nature, 330:537 (1987)) has a single extracellular domain (xcx9c28 kD), a transmembrane segment, and an intracellular domain (xcx9c30 kD) that is not homologous to any known tyrosine kinase or other protein. Nonetheless, the extracellular portion of the hGH receptor is structurally related to the extracellular domains of the prolactin receptor (Boutin, J. M. et al., Cell 69:(1988)) and broadly to at least eight other cytokine and related receptors. hGHbp expressed in Escherichia coli has been secreted in tens of milligrams per liter (Fuh, G., et al., J. Biol. Chem., 265:3111-3115 (1990)). The highly purified hGHbp retains the same specificity and high affinity for hGH (KD xe2x88x920.4 nM) as compared to the natural hGHbp found in serum.
hGH is a member of an homologous hormone family that includes placental lactogens, prolactins, and other genetic and species variants of growth hormone (Nicoll, C. S., et al. Endocrine Reviews,7:169(1986). hGH is unusual among these in that it exhibits broad species specificity and binds to either the cloned somatogenic (Leung, D. W. et al. [1987] Nature 330: 537) or prolactin receptor (Boutin, J. M., et al. Cell, 53:69). The cloned gene for hGH has been expressed in a secreted form in Eschericha coli (Chang, C. N., et al., Gene 55:189 [1987]) and its DNA and amino acid sequence has been reported (Goeddel, et al. Nature,281:544 [1979); Gray, et al., Gene 39: 247[1985]). The three-dimensional structure of hGH has not previously been available. However, the three-dimensional folding pattern for porcine growth hormone (pGH) has been reported at moderate resolution and refinement (Abdel-Meguid, S. S. et al. Proc. Natl. Acad. Sci. USA, 84:6434 [1987]). hGH receptor and antibody binding sites have been identified by homolog-scanning mutagenesis (Cunningham, et al, Science 243:1330, 1989). GH with N-terminal amino acids deleted or varied are known. See Gertler, et al., Endocrinology 118:720 (1986), Ashkenazi, et al., Endocrinology 121:414 (1987), Binder, Mol. Endo.,7:1060-1068 (1990), and WO 90/05185. Antagonist variants of hGH are described by Chen, et al., Mol. Endo., 5(10):1845 (1991) and literature set forth in the bibliography thereof; and WO 91/05853. hGH variants are disclosed by Cunningham, et al., Science, 244:1081 (1989) and Science 243:1330-1336 (1989).
Since the mode of interaction of many polypeptide ligands with their receptors has remained uncertain it has been difficult to engineer amino acid sequence variants of such ligands to achieve desired properties. Essentially, the art has introduced variation at random, perhaps in some cases with guidance from homology analyses to similarly-acting ligands or animal analogues, or from analysis of fragments, e.g., trypsin digest fragments. Then the art has screened the candidates for the desired activity, e.g., agonist or antagonist activity. The screening methods have been tedious and expensive, e.g., the use of transgenic animals (WO 91/05853). Methods are needed for improving the efficiency of selection of candidates. In particular, methods are needed for focusing on candidates likely to be either antagonists or agonists. Antagonists are substances that suppress, inhibit or interfere with the biological activity of a native ligand, while agonists exhibit greater activity per se than the native ligand.
It therefore is an object of this invention to provide improved methods for the efficient selection of agonist or antagonist polypeptide ligands.
It is another object herein to provide a method for detecting ligands that form sequential 1:2 complexes with their receptors.
Another object herein is to assay candidate substances for their ability to interfere with or promote the formation of such 1:2 ligand-receptor complexes.
An additional object is to provide amino acid sequence variants of polypeptide ligands that are capable of acting as agonists or antagonists.
Other objects, features and characteristics of the present invention will become more apparent upon consideration of the following description and the appended claims.
We have unexpectedly found that growth hormones and the class of conformational ligands to which they belong are capable of forming 1:2 complexes with their receptor in which a first ligand site, site 1, binds to one receptor and then a second ligand site, site 2, binds to another molecule of receptor, thereby yielding a 1:2 complex. The ligands to which this invention are applicable are monomeric ligands containing 4 amphipathic antiparallel alpha-helical domains separated and terminated at both ends by non-helical amino acid sequences. It is now possible by analogy to our work with growth hormone, prolactin and placental lactogen to efficiently design agonist or antagonist amino acid sequence variants of such ligands by introducing amino acid sequence variation into sites 1 and/or 2 as will be more fully described below.
The two-site complex formation assay is used to screen for substances which are ligand agonists or antagonists. Such substances are essentially unlimited and include organic, non-proteinaceous compounds as well as amino acid sequence variants of the ligands and binding protein or receptor variants.
New amino acid sequence variants of such alpha helical ligands also are described. In particular, antagonists for polypeptide ligands are provided which comprise an amino acid sequence mutation in site 2 which reduces or eliminates the affinity of the ligand for receptor at site 2. Ideally, the ligand antagonist analogue will have low or no affinity for receptor at site 2 and and will have elevated affinity for receptor at site 1.
Also provided herein are agonist ligand amino acid sequence variants having mutations at sites 1 and/or 2 which increase the ligand affinity for one or both sites. In prefered embodiments, the rate constants for both sites are selected such that the average residence time of the ligand in the dimer complex is greater than or equal to the time required for the complex to effect the desired cellular response. Polypeptide agonist variants of the ligand are identified by a method comprising (a) introducing a mutation into the ligand to produce an agonist candidate, (b) determining the affinity with which the candidate binds to the receptor through its first ligand site, (c), determining the affinity with which the candidate binds to the receptor through its second ligand site, and (d) selecting the candidate as an agonist if it binds at one or both of the first and second sites with greater affinity than the native ligand.
In accordance with this invention a method is provided for detecting an agonist or antagonist candidate for a polypeptide ligand, which ligand normally binds in sequential order first to a receptor polypeptide through a first ligand site and secondly to a second copy of the receptor polypeptide through a second ligand site different from the first site, comprising determining the effect of the candidate on the affinity of the polypeptide ligand for receptor at the ligand""s second receptor binding site. Site 1 interactions are determined by immunoprecipitation using a site-2 blocking antibody such as Mab5 as described infra. Alternatively, the amount of wild type ligand that substantially forms only a 1:1 complex with receptor is determined and then the ability of the candidate to compete with native ligand for receptor at that proportion is determined. Site 2 interactions are assayed by following the ability of the candidate to form the ternary complex.
Where the candidate is a polypeptide analogue of the ligand then one positively correlates an absence of binding of the analogue at site 2 with antagonist activity. The ability to bind with greater affinity than native ligand to receptor site 2 is correlated with agonist activity. Antagonist and agonist activity are both positively correlated with the ability of the candidate to bind at site 1 with greater affinity than native ligand. Small molecule or other non-analogous candidates are assayed for their ability to promote or suppress binding of native ligand to sites 1 and/or 2. Antagonists are screened for their ability to interfere with native ligand-receptor binding at site 2 and/or site 1, but preferably site 2. This permits the identification of antagonists that do not suppress ligand receptor binding at site 1 but which do interfere with site 2 binding, using for example as a positive control a site 2 disabled variant of the ligand.
The effect of the candidate can be measured in the presence of the native polypeptide ligand or in comparison to the activity of the native polypeptide ligand. In the first alternative, the effect of the candidate on receptor interactions by the wild type ligand is measured. In the second the activity of the wild type ligand is used as a positive control and the receptor binding characteristics of the candidate (usually an amino acid sequence variant of the ligand) are measured without the presence of the wild type ligand. In general, however, the assays for agonist or antagonist candidates are best conducted as competition-type assays in the presence of wild type ligand.
We also have determined that selected antibodies capable of binding the GH receptor act as antagonists or agonists of GH. Accordingly, methods are provided for the antagonism or agonism of GH in the therapy of growth hormone deficiency or excess.